Inflammatory Cytokine Production of LPS-stimulated Macrophage Exposed to Ni Ions

Objective: Ni ions are known to cause cellular damage and allergic reaction, in which inflammatory cytokines play significant roles. The purpose of this study was to clarify the effects of Ni²⁺ ion concentrations on the production of three inflammatory cytokines of lipopolysaccharide (LPS)-stimulated mouse macrophage-like RAW264 cells. Methods: RAW264 cells with and without 10 µg/mL LPS were cultured for 2 days in minimum essential medium supplemented with 10% fetal bovine serum and Ni²⁺ ion concentrations of 0, 50, 200, 500 and 1000 µmol/L, respectively. The DNA amount in cell lysate was measured with dye (170-2480, Biorad) and fluorometer. Three inflammatory cytokines (TNF-α, IL-1β and IL-6) in the culture supernatant were measured using ELISA kits (Pierce Endogen). Results: (1) Without LPS, DNA amounts of RAW264 declined with incrementing the Ni²⁺ ion concentration. When LPS-stimulated, DNA amounts of RAW264 remained constant. (2) Without LPS, very minute TNF-α and IL-6 were produced, proportionally to the Ni²⁺ ion concentration, and the production of IL-1β was absent. With LPS, it was confirmed that LPS(+) Ni²⁺ ion-free RAW264 secreted abundant three cytokines, 100 times more than LPS(-) Ni²⁺ ion-free RAW264. Addition of Ni²⁺ ions to LPS (+) RAW264 caused different three cytokine production results. TNF-α was increased with incrementing the Ni²⁺ ion concentration from 0 to 200 µmol/L, then declined to 1000 µmol/L. IL-β was increased with incrementing the Ni²⁺ ion concentration from 0 to 500 µmol/L, then declined to 1000 µmol/L. IL-6 peaked at the Ni²⁺ ion concentrations from 0 to 200 µmol/L, then gradually declined to 1000 µmol/L. Conclusion: It was confirmed that low dose of Ni²⁺ ions tended to accelerate inflammatory cytokine productions, especially that of IL-1β, of LPS-stimulated macrophages. It was speculated that Ni²⁺ ions might positively interact with a Nf-κB signaling pathway, causing initial increase in cytokine productions.